Double-strand breaks in chromosomal DMAare a constant threat to all organisms, and unrepaired or misrepaired lesions can lead to deleterious genomic rearrangements or cell death. The cellular response to DNA double-strand breaks involves a rapid mobilization of DMA repair factors as well as signaling molecules to the damage sites, which initiates DNA repair and triggers cell cycle arrest. These responses to DNA breaks are critical for the maintenance of genomic stability, and loss of the cellular components of these pathways facilitates the genomic mutations and rearrangementsthat can lead to cancer in humans. The Mre11/Rad50/Nbs1(Xrs2) (M/R/N(X)) complex plays a central role in these events by initiating DNA double- strand break repair as well as recruiting and activating signaling molecules. This proposal addresses the biochemical activities of the M/R/N(X) complex with the overall goal of understanding how these activities are related to functions of the complex in cells at sites of DNA damage. In previous work we used recombinant human M/R/N complex to elucidate the enzymatic activities of the complex on model DNA substrates and on the activities of ATM, the primary transducer of the DNA damage signal that originates from DNA double- strand breaks. In the current proposal, this biochemical approach is extended to also include the S. cerevisiae M/R/X and P. furiosus M/R complexes in order to dissect the conservedcatalytic activities of this enzyme and to efficiently isolate mutants that delineate key functions of each component. With this strategy we will address the substrate specificity of M/R/N(X) nuclease activity on hairpin structures and on covalent protein-DNA conjugates in vitro. We will also determine the specific roles of the RadSO catalytic domain, coiled-coil, and zinc hook in M/R/N(X)-DNA interactions in vitro as well as in vivo. These experiments will bridge the gap between our knowledge of the biochemistry of this complex and observations of the biological consequences of M/R/N(X) mutations in yeast and in mammalian cells. By characterizing the basic mechanisms of enzymes involved in DNA repair and DNA damage signaling, we can elucidate the normal cellular responses to DNA lesions. This approach is essential for an understanding of the earliest events in cancer progression which involve spontaneous or inherited defects in these pathways, and provides the molecular tools for subsequent diagnostic and therapeutic reagents.